Various patents and scientific articles are referred to throughout the specification. These publications are incorporated by reference herein to describe the state of the art to which this invention pertains.
Breast cancer has caused the death of a quarter of a million women worldwide for many years and has been estimated to be the leading cause of death in women aged between 35 to 54, being second only to cardiovascular diseases in women aged over 55 (W. P. D. Logan, Cancer of the female breast. International mortality trends. W. H. O. Stat. Rep. 28:232, 1975).
Breast cancer accounts for 27% of all malignancies around the world. Despite improvements in early detection of this disease, its incidence is increasing and its mortality rate has not significantly decreased. There is a well-known association between early full term pregnancy and a reduction in the lifetime risk of developing breast cancer, however the mechanisms mediating this protective effect have not been elucidated.
In the rat model, completion of a full term pregnancy prior to carcinogen administration inhibits tumor production. Maximal mammary tumor incidence occurs when the carcinogen is administered to young rats (Russo and Russo, 1987, Lab. Invest. 57:112–137; Russo and Russo, 1978, J. Natl. Cancer Inst. 61:1439–1442; Russo et al., 1979, Am. J. Pathol. 96:721–734). Tumor incidence decreases significantly, or becomes almost completely abolished when the carcinogen is administered to parous rats between 3 and 9 weeks post-delivery with or without lactation (Russo and Russo, 1980, Am. J. pathol. 100:497–511; Russo and Russo, 1980, Cancer Res. 40:2677–2687; Russo and Russo, 1982, Internat. Res. Com. (IRCS) 10:935–945; Russo and Russo, 1993, European J. Cancer Prevention 2:101–111).
The role played by endocrine treatment in breast cancer was discovered in 1896, when Bateson observed that breast cancer in pre-menopausal women undergoes remission after oophorectomy. This finding, subsequently confirmed by other scientists, supported the evidence that at least some breast tumors are directly dependent on hormones for their growth and created interest in the therapeutic approach of endocrine organ ablation for the purpose of removing the endogenous source of hormones.
As drugs specifically antagonizing the estrogen action were discovered, they became an attractive alternative to surgical ablation. Several anti-estrogen compounds have been tested in pre- and post-menopausal women in phase I and II clinical trials. So far, Tamoxifen has proved to be the drug best approaching the effectiveness of surgical endocrine therapy and the one that is substantially free from serious side effects. A comprehensive review of the therapeutic efficacy of antiestrogens in the treatment of breast cancer can be found in Legha & Carter, Antiestrogens in the treatment of breast cancer. (Cancer Treat. Rev. 3:205, 1976.). Another review more specifically related to clinical experience with Tamoxifen is that of Paterson et al, A review of the International clinical experience with Tamoxifen. (Jpn. J. Cancer Clin. 11 (Suppl.): 157, 1981).
Approximately one-third of women with breast cancer respond to antiestrogen-based hormonal therapy, while an increase up to 70% of response is expected in patients with receptor-rich tumors. In fact, estrogen receptor (ER) status has been demonstrated to be predictive of response in breast cancer patients (Allegra, J. C., Reviews on Endocrine related cancer. Paterson A H G, Lees A W eds Suppl. 14:115, 1984).
Chorionic gonadotropin is a glycoprotein hormone composed of two non-covalently linked (α and β) subunits. (Labrie, Glycoprotein hormones: gonadotropins and thyrotropin. In: Hormones—From Molecules to Disease. Beanlien E E and Kelly P A—Chapman and Hall, New York and London, pp 257–275, 1990). It is synthesized early in pregnancy by the developing embryo and throughout the gestational process by the syncytiotrophoblast of the placenta, and it is secreted in urine.
Human chorionic gonadotropin is obtained from the urine of pregnant women for both experimental and clinical uses. The hormone can also be prepared via the recombinant route (WO 85/01959) The main known function of hCG is the stimulation of gonadal steroid hormone production through its interaction with the LH/CG receptor, which is present in the granulosa cells of the ovary in the female and in the testicular Leydig cells in the male.
Recent studies have suggested that urinary hCG is a potent preventive agent that inhibits chemically-induced mammary tumorigenesis through the induction of differentiation. (Russo et al., J. Natl. Cancer Inst. 82:1286–1289, 1990). Additional experiments indicated that hCG treatment of rats after exposure to carcinogens also protected them from tumor development (Russo et al., Br. J. Cancer 62: 2343–2347, 1990). HCG also inhibits the proliferation of normal and neoplastic human breast epithelial cells (Alvarado et al., In Vitro 30A: 4–8, 1994). It has also been found that urinary hCG from various sources has an inhibitory effect on neoplastic cell lines from various organs or systems (Gill et al., J. Natl Cancer Inst. 89: 1797–1802, 1997; Albini et al., AIDS 11: 713–721, 1997; Mgbonyebi et al., Proc. Annu. Meet. A, Soc. Cancer Res. 38, PP. A1977, XP002109660, 1997).
International Patent Application WO 97/49432 describes the induction of cell death of breast cancer cells in vitro by treatment of the cells with urinary hCG. The same effect was observed with various fractions and the hCG β-subunit.
Two further studies, both performed in a rat model, showed that urinary hCG has a preventive effect against carcinogen-induced rat mammary tumors (Srivastava et al., Carcinogenesis 18: 1799–1808, 1997 and You et al., Cancer Research 58/7: 1498–1502, 1998). Rats were pre-treated with daily doses of urinary hCG for several weeks before tumor induction and sacrificed at various time points after continued hCG treatment. In the pre-treated rats, the incidence of carcinoma-induced mammary tumors was reduced.
Women who had undergone urinary hCG treatment for infertility or weight loss also were observed to have a reduced incidence of breast cancer (Bernstein et al., Cancer Epidemiol., Biomarkers and Prev. 4: 437–440, 1995). These observations were only inferential, however, and studies directed toward measuring the effect of hCG in the treatment of human breast cancer heretofore have not been reported. From the rat model, only a protective effect of urinary hCG on the development of cancer has been deduced when the hCG was administered very close to initiation of carcinogenesis. However, in humans, breast cancer is usually diagnosed only after it is already established as a palpable nodule, or detectable in diagnostic mammography.
U.S. Pat. No. 5,700,781 (Harris, 1997), U.S. Pat. Nos. 5,677,275 and 5,877,148 (Lunardi-Iskandar et al., 1997, 1999), as well as International Patent Application WO96/04008 disclose methods for treating cancers like Kaposi's sarcoma, involving the administration of urinary hCG. For Kaposi's sarcoma, which is believed to be of endothelial origin, the hCG seemed to have an anti-proliferative effect when injected into mice having established, metastatic Kaposi's sarcoma.
Recently, the anti-tumor effects of hCG were questioned by a series of experiments reported by the group of Robert Gallo (Lunardi-Iskandar et al., Nature Med. 4: 428–434, 1998 and comments at pages 370 and 390–391 of the same issue). These experiments convincingly showed that the anti-Kaposi's Sarcoma (KS) effect of hCG preparations are not due to the hCG itself, but instead to an unidentified urinary factor, referred to as HAF (hCG-associated factor). A number of commercially available hCG preparations (clinical grade and purified), as well as subunits, fragments and recombinant hCG, were tested for their inhibitory activity on neoplastic KS cells in vitro and in in vivo animal models. Neither purified urinary nor purified recombinant hCG, subunits or fractions of hCG had any effect in the in vivo animal model or in vitro assay, respectively.
The fact that early pregnancy of mice leads to KS tumor regression even though mice do not produce hCG at all further supports these findings.
Taken together, evidence accumulated that the beneficial effects of hCG in treating cancer were just an artifact of insufficient protein purification, the actual anti-tumor agent being an as-yet unidentified factor associated with early pregnancy. The results obtained with recombinant hCG, together with the fact that the source of the hCG-associated factor “HAF” was first-trimester pregnancy human urine, ruled out the possibility of obtaining any anti-tumor activity from recombinant hCG preparations.